Gaucher Disease: We have continued our investigations on the pathogenesis of the neuronopathic forms of Gaucher disease. In addition to the accumulation of glucocerebroside in the brain of patients with this phenotype, significantly increased quantities of glucosylsphingosine (GlcSph) occur in the CNS. We demonstrated that GlcSph is highly toxic to cultured neuronal cells in concentrations that are present in the brain of patients with both the acute and chronic neuronopathic forms of Gaucher disease. We deduce that GlcSph plays a major role in neuronal dysfunction and destruction in patients with neuronopathic Gaucher disease. We investigated the biosynthesis of GlcSph and found an enzyme in brain that catalyzes its formation. We have identified seven compounds that inhibit the enzymatic synthesis of GlcSph. We shall determine which of these inhibitors is the most effective, its Ki and its ability to cross the blood-brain barrier. If the identified compound reaches the brain in an effective concentration and it is not toxic at this level, we shall conduct a clinical trial with it to attempt to improve the debilitating clinical course of patients with neuronopathic Gaucher disease. We have also undertaken several strategies to improve on and make enzyme replacement therapy less costly. To accomplish these goals, we have introduced conservative substitutions in the amino acid sequence of glucocerebrosidase, the enzyme that is deficient in Gaucher disease. We expect that such modifications will increase the stability of the enzyme thereby reducing the quantity that is required to produce a therapeutic benefit in patients with this disorder. We are also exploring strategies to improve the biodistribution of glucocerebrosidase that include intracellular transport and membrane recognition domains that function in endosomal segregation and delivery of enzymes to lysosomes. In addition, we are developing methods to improve gene therapy for Gaucher disease using lentiviral vector constructs. The first of these has been shown to significantly increase the level of glucocerebrosidase activity in multiple organs and tissues when injected into experimental animals. Moreover, it very effectively transduces bone-marrow stem and progenitor cells ex vivo. It is anticipated that these cells may be appropriate for gene therapy trials in patients with Gaucher disease since, if successful, bone marrow transplantation has been shown to cure patients with type 1 (non-neuronopathic) Gaucher disease. Fabry disease: One of the important aspects concerning the pathogenesis of metabolic storage disorders is the relationship between the reduction of catalytic activity of a particular enyzme and the extent of accumulation of its substrate. We have initiated an investigation to determine the threshold level of alpha-galactosidase A, the enzyme that is deficient in Fabry disease, and the accumulation of the offending lipid globotriaosylceramide (Gb3). This information is potentailly very important in order to estimate the dose of exogenous enzyme that is required for effective enzyme replacement therapy. It should also provide insight into predicting the extent of clinical manifestations in heterozygous female carriers of Fabry disease who exhibit widely varying levels of residual alpha-galactosidase A activity. In addition, we have negotiated a Cooperative Research and Development Agreement to explore the use of a small molecular weight active site-specific chaperone that has been shown to increase the catalytic activity caused by a number of mutations of alpha-galactosidase A. After Phase 1 safety and dose-resonse trials have been completed, we shall examine the clinical effectiveness of active site-specific chaperone therapy in appropriate patients with Fabry disease. Delivery of Genes to the Central Nervous System: We have incorporated the human glucocerebrosidase (GC) gene into a lentiviral construct and demonstrated that it functions very well in vitro. Delivery of the GC gene to the CNS is significantly enhanced if intravenous injection of this vector is accompanied by intraperitoneal administration of a hypertonic solution of mannitol. Incorporation of herpes simplex virus type 1 tegument protein VP-22 into the GC lentiviral vector facilitates the intercellular delivery of GC from transduced cells to non-infected cells. A number of collaborative studies are underway with intramural and extramural scientists who are using the vector systems we developed. One of these is an investigation of the role of G-proteins on the mode of action of hallucinogenic drugs. In another study, the incorporation of the gene for neuron-specific enolase into the lentifival CG vector has been shown to greatly increase the transduction of CA1 pyramidal neurons in organotypic slice cultures. This vector will be used in attempts to generate transgenic rats. In addition, the potential of lentiviral-mediated RNA interference will be evaluated. The findings from this investigation will serve as a basis to knock-down physiologically important genes in brain slice cultures and by stereotactic injection into the brain in vivo. A further investigation is underway with these vectors to determine the function of Numb and Numblike genes on the regulation of neuronal projections and fate of cells in the developing brain. Mucolipidosis IV (MLIV): We discovered that MCOLN1, the gene that is mutated in patients with this disorder, codes for a protein called mucolipin that is a member of the TRP family of proteins. We have obtained evidence that MLIV is a channelopathy. We have achieved selective silencing of mucolipin RNA and blocked the expression of the MLIV gene in a continuous human parietal cell line that will serve as a model of the loss of gastric acid production, a hallmark of MLIV. We have also prepared a targeted gene construct to knock out MCOLN1. Investigations are underway to create a MLIV knock-out mouse that will be exceptionally valuable for pathogenic and therapeutic investigations.